Acoustic modulation of mechanosensitive genes and adipocyte differentiation

Eukaryotic cells are equipped with multiple mechanosensing systems and perceive wide range of mechanical stimuli from the environment. However, cell-level responses against acoustic waves, which transmits feeble but highly frequent physical perturbation, largely remains uninvestigated, especially with regard to audible range of sound. Here we investigate the effect of acoustic stimulation on gene expression profiles of mammalian cultured cells. A direct sound emission system was set up using a vibrational transducer to directly generate acoustic waves in culture medium. A custom-made vibrating plate made of PEEK (poly ether ether ketone) plastic was used as a diaphragm. A set of sound patterns including single-frequency sound and white noise were generated by NCH Tone Generator software. Sound intensity was directly measured by recording it in water using a hydrophone and the pressure level was calculated. C2C12 myoblasts cultured in a plastic dish with approximately 50% confluency were subjected to acoustic stimulation. 440 Hz and 14k Hz single-frequency sine wave sound were selected as representatives of low and high audible frequencies, and white noise was selected as a random noise pattern. 2 and 24 hours after continuous emission of these sound at 100 Pa, total RNA was extracted and subjected to the gene expression profiling analysis by RNA-sequencing technique. Total 42 early- and 145 late-response genes were identified as sound-sensitive genes in 2 and 24 hours stimulation, respectively. Gene annotation analyses revealed that in addition to the known mechanosensitive activities such as fluid shear stress response, cell migration, cell adhesion and blood vessel development, variety of pathways and processes were identified to be affected by acoustic stimulation. Overall design: C2C12 cells were subjected to acoustic stimulation by a custom-made direct sound emission system using vibrational transducer and a diaphragm. Low frequency (440 Hz), high frequency (14 kHz), and white noise were emitted to the cells at 100 Pa intensity, for 2 and 24 hours.